Detection and measurement of human cardiac troponin I (cTnI) in serum has been proposed as a sensitive early indicator of myocardial infarction (Larue et al., (1992), Molec. Immunol. v. 29, pp. 271-278 and (1993), Clin. Chem. v. 39, pp. 972-979; Bodor et al., (1992), Clin. Chem., v. 38, pp. 2203-2214). For reproducible and reliable clinical testing, stable calibration standards and quality control standards are required. Such standards require pure and stable preparations of intact human cardiac troponin I.
cDNA coding for human cTnI has been cloned and sequenced (Vallins et al., (1990), FEBS Letters, v. 270, pp. 57-61; Hunkeler et al., (1991), Circ. Res., v. 69, pp. 1409-1414). The cDNA indicates an amino acid sequence of 210 amino acids or a molecular weight of approximately 28 kDa. Purified preparation of cTnI, however, have been reported to have molecular weights of 22.5 kDa (Lame et al. (1992); Bodor et al. (1992)), suggesting a loss of pan of the molecule.
Furthermore, previously described methods for the isolation and purification of cTnI (Tsukui et al., (1973), J. Biochem. v. 73, pp. 1119-1121; Cummins et al., (1978), Biocem. J. v. 171, pp. 251-259; Syska et al. (1974), FEBS Lett. v. 40, pp. 253-257) yielded preparations which were unstable and subject to considerable degradation on storage.
Protease inhibitors have been suggested for use in the purification of cardiac troponins, but generally only one or two inhibitors were used (Beier et al., (1988), Eur. J. Biochem., v. 176, pp. 327-334; Jin et al., (1988), J. Biol. Chem. v. 263, p. 7309; Stull et al., (1977), J. Biol. Chem. v. 252, pp. 851-857).
Beier et al., (1988), used phenylmethylsulphonyl fluoride (PhMeSO.sub.2 F) and benzamidine as inhibitors during the extraction and isolation of troponin from bovine heart tissue. This purification method involved an extraction with high salt (LiCl) followed by ammonium sulfate ((NH.sub.4).sub.2 SO.sub.4) precipitation and DEAE-cellulose column chromatography. Jin et al., (1988), used phenylmethanesulphonyl fluoride (PMSF) in their extraction procedure for the purification of cardiac troponin T from beef heart. This purification method included a 60.degree. C. treatment of a high salt (KCl) extract, ammonium sulfate fractionation and DEAE-cellulose column chromatography. Stull et al., (1977), also used PMSF in their extraction procedure for isolating troponin complex containing both troponin C, troponin I, and troponin T from beef hearts. This purification procedure involved ethanol extraction, ether wash, high salt treatment with 1 M KCl, ammonium surfate fractionation and column chromatography with a Bio-Gel sepharose column and hydroxylapatite chromatography.
There remains a need for a method for purifying cTnI which will protect the integrity of the molecule and yield cTnI preparations which can meet the stringent stability requirements for clinical assay standards and calibrators.